Project Summary/Abstract The objectives of this project are to reprogram primary T cells as a means of eliminating cancer cells?a major goal of biomedical research as mandated by the National Institutes of Health. Using new genetic tools applied in novel ways, we will overcome present limitations to cancer immunotherapy by produc- ing T cells that have been relieved of multiple inhibitory feedback mechanisms. However, the more ef- fective the T cell population in eliminating cancerous cells, the more likely they are to cause inflammato- ry immunopathology. Thus, an important requirement for this reprogramming is to provide a provision for their elimination. The means for producing such T cells relies on a recently developed autocatalytic gene conversion method based on the Crispr/Cas9 system and known as the mutagenic chain reaction (MCR). There are two primary advantages to our integrated system for reprogramming T-cells. First, our meth- od should be substantially more efficient in generating biallelic insertions that can render cells defective for multiple genes. Second, because MCR vectors integrate larger inserts into the genome with high effi- ciency and fidelity, we will be able to introduce a multifunctional cassette of genetic elements (e.g., sgR- NAs targeting multiple inhibitory pathways and an inducible suicide module). Combined, these two fea- tures will make it possible to reprogram effector T cells in a single round of ex vivo treatment reducing the number of cell divisions that take place in culture and minimizing the time from cell retrieval to rein- fusion of tumor-fighting T cells. The anti-tumor efficacy of reprogrammed T cells will be studied in models of solid and metastatic melanoma to identify the optimal combination of targeted inhibitory pathways. Initial experiments will take advantage of mouse models, with experiments progressing to the reprogramming of cultured human T cells. These techniques will enable a generation of novel therapies to treat cancer, but also chronic infectious diseases, autoimmunity, and allergic hypersensitivity diseases.